Part:BBa_K864016

Low copy BioBrick temperature sensitive standard vector

pSB8A15 is a BioBrick standard vector with low copy pSC101ts replication origin BBa_K864051 (~5 copies per cell) and ampicillin antibiotic resistance marker, especially usable for Lambda Red recombineering in E coli. The pSC101ts is a temperature sensitive pSC101 replication origin and propagates at 30ºC but not at at 42ºC [1]. The backbone sequence is based on pSB3T5, but the E coli His operon terminator BBa_B0053 has been replaced with the late terminator of the Salmonella phage P22.

Usage and Biology

This part is available as pSB8A15-pUCori-redq. This facilitates fast growth at 37° C, high plasmid yields and fast red color expression on transformation plates.

FLP recombinase

The pSB8X15 series are especially suitable for carrying systems where a temporary expression is desired, such as the Lambda Red recombineering system or recombinases like Cre or FLP, since the plasmids can be removed afterwards by incubation at 42°C.

Origin switching

Easy ori replacement is possible in the pSB8x15 backbones. The plasmid can have the pSC101ts ori cut out with NheI and MluI and another ori ligated in. To remove any religated pSC101ts, the transformants can be grown at 42° C.

More reliable recombineering

A problem when doing recombineering based chromosomal intergration is that some clones may take up the plasmid template instead of recombining the PCR fragment into the chromosome. When doing recombineering with pSB8x15 backbones, any such clones can be removed by incubation at 42° C.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 3618
  Illegal NheI site found at 2425
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal NotI site found at 9
  Illegal NotI site found at 3624
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 3618
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found at 3618
  Illegal suffix found at 2
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found at 3618
  Plasmid lacks a suffix.
  Illegal XbaI site found at 3633
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal NgoMIV site found at 2319
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  

References

[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC235445/] T Hashimoto-Gotoh, M Sekiguchi: "Mutations of temperature sensitivity in R plasmid pSC101" J. Bacteriol. 131.2 (1977) 405-412

[http://dx.doi.org/10.1016/0022-2836(84)90352-8] K.A. Armstrong, R. Acosta, E. Ledner, Y. Machida, M. Pancotto, M. McCormick, H. Ohtsubo, E. Ohtsubo: "A 37x10(3) molecular weight plasmid-encoded protein is required for replication and copy number control in the plasmid pSC101 and its temperature-sensitive derivative pHS1" J. Mol. Biol., 175 (1984), 331€“348